Abstract

Background

The spatial organization of the genome is being evaluated as a novel indicator of
toxicity in conjunction with drug-induced global DNA hypomethylation and concurrent
chromatin reorganization. 3D quantitative DNA methylation imaging (3D-qDMI) was applied
as a cell-by-cell high-throughput approach to investigate this matter by assessing
genome topology through represented immunofluorescent nuclear distribution patterns
of 5-methylcytosine (MeC) and global DNA (4,6-diamidino-2-phenylindole = DAPI) in
labeled nuclei.

Methods

Differential progression of global DNA hypomethylation was studied by comparatively
dosing zebularine (ZEB) and 5-azacytidine (AZA). Treated and untreated (control) human
prostate and liver cancer cells were subjected to confocal scanning microscopy and
dedicated 3D image analysis for the following features: differential nuclear MeC/DAPI
load and codistribution patterns, cell similarity based on these patterns, and corresponding
differences in the topology of low-intensity MeC (LIM) and low in intensity DAPI (LID)
sites.

Results

Both agents generated a high fraction of similar MeC phenotypes across applied concentrations.
ZEB exerted similar effects at 10–100-fold higher drug concentrations than its AZA
analogue: concentration-dependent progression of global cytosine demethylation, validated
by measuring differential MeC levels in repeat sequences using MethyLight, and the
concurrent increase in nuclear LIM densities correlated with cellular growth reduction
and cytotoxicity.

Conclusions

3D-qDMI demonstrated the capability of quantitating dose-dependent drug-induced spatial
progression of DNA demethylation in cell nuclei, independent from interphase cell-cycle
stages and in conjunction with cytotoxicity. The results support the notion of DNA
methylation topology being considered as a potential indicator of causal impacts on
chromatin distribution with a conceivable application in epigenetic drug toxicology.